Culture vessel for growing or culturing cells, cellular aggregates, tissues and organoids and methods for using same

ABSTRACT

A bioreactor system for culturing cells, cellular aggregates, tissues and organoids and methods of using same includes a cylindrical reactor vessel having an unobstructed longitudinal axis that is rotated about that horizontal axis to suspend cells and/or tissue in a culture medium. Filters are used within the vessel to retain the cells and/or tissues within the culture chamber and may be also used to subdivide the culture chamber. A pump maintains a flow of oxygen rich culture medium through the vessel to sustain cell respiration. A gas exchange device maintains desired gas concentrations in the medium and the entire system is operated within an enlarged incubator to maintain appropriate temperatures. Access ports on the vessel are used to introduce various materials into the culture chamber and to remove bubbles that may form in the medium. Sensors and a microprocessor unit monitor operational conditions and direct adjustments to maintain desired conditions. 
     Several methods of operating the bioreactor system are also disclosed wherein the vessel is used to culture cells, to grow three dimensional cellular aggregates and tissues, to filter waste materials and toxins from fluids, to commercially produce various biological materials from cells and/or tissue, and as a diagnostic or research tool.

This application is a division of U.S. Ser. No. 09/109,492, filed Jul.2, 1998, by Charles Daniel Anderson and Charlie W. Dodd entitled"Culture Vessel for Growing or Culturing Cells, Cellular Aggregates,Tissues and for Using Same", now U.S. Pat. No. 5,989,913.

FIELD OF THE INVENTION

This invention relates to an apparatus and method of use for a newculture vessel or bioreactor. The vessel is useful for growing cellcultures growing three dimensional cellular aggregates or tissues, forcarrying out various cellular processes with tissues and/or organoidsand for diagnostic testing and research.

BACKGROUND OF THE INVENTION

Bacterial cell culture processes have been developed for the growth ofsingle cell bacteria, yeast and molds which can be characterized asencased with a tough cell wall. Large scale culture of bacterial typecells is highly developed and such culture techniques are less demandingand are not as difficult to cultivate as mammalian cells. Bacterialcells can be grown in large volumes of liquid medium and can bevigorously agitated without any significant damage.

Mammalian cell culture and tissue generation, however, is much morecomplex because such cells are more delicate and have a more complexnutrient requirement for development. Mammalian cells cannot withstandexcessive turbulent action without damage to the cells and must beprovided with a complex nutrient medium to support growth. Therefore,bioreactors with internal moving parts or obstructions will subjectmammalian cells to high fluid shearing forces that will damage thecells. In addition, bioreactors that utilize mechanical parts, air orfluid movement as a lift mechanism to achieve particle suspension willlikewise cause damage to growing cells and tissues due to fluid shear.

A primary use of bioreactors is in research where large numbers of cellsare grown to refine the minute quantities of an active material (e.g.proteins) that the cells might secrete. Another use of bioreactors isthe scale-up of laboratory cell culture processes for commercialpurposes to mass produce the active proteins made by geneticallyengineered cells and tissues. Because of the need to culture mammaliancells in the laboratory in large quantities, bioreactors and culturingvessels have become an important tool in research and production ofcells that produce active proteins.

A current problem in tissue culturing technology is the unavailabilityof an effective bioreactor for the in vitro cultivation of cells andexplants that allows easy access to the materials contained in thevessel. Several devices presently on the market have been used with onlylimited success since each has limitations which restrict usefulness andversatility. Further, no bioreactor or culture vessel is known that willallow for the unimpeded growth of three dimensional cellular aggregatesor tissues.

Cell culturing devices range upward in complexity from the petri dish tosophisticated computer controlled bioreactors. In the past,manufacturers have promoted various technologies to culture cells in thelaboratory. For instance, simple adaptations of fermentors or stirredtanks used for the culture of bacteria, were marketed previously as theanswer to culturing delicate mammalian cells. One of the principalfactors limiting the performance of these systems is their inability tominimize turbulence due to stirring, i.e., shear due to fluid flow, andhence preventing free form association of cells in three dimensions.

PRIOR ART

A variety of methods and devices have been developed around the conceptof horizontally rotating vessels for the suspension of solids in liquidmedia. Examples of such devices include bioreactors for cell culture asshown in FIGS. 1-3. Patents on such devices include, U.S. Pat. No.5,155,035, issued to Schwarz et al., and entitled "Method For CulturingMammalian Cells In A Perfused Bioreactor"; U.S. Pat. No. 5,155,034,issued to Wolf et al., and entitled "Three Dimensional Cell To TissueAssembly Process"; U.S. Pat. No. 5,153,133,issued to Schwarz et al., andentitled "Method For Culturing Mammalian Cells In A Horizontally RotatedBioreactor"; U.S. Pat. No. 5,153,131, issued to Wolf et al., andentitled "High Aspect Vessel And Method Of Use"; U.S. Pat. No.5,026,650, issued to Schwarz et al., and entitled "Horizontally RotatedCell Culture System With A Coaxial Tubular Oxygenator"; and U.S. Pat.No. 4,988,623, issued to Schwarz et al., and entitled "RotatingBio-Reactor Cell Culture Apparatus." These patents are incorporatedherein by reference as if set out fully verbatim. U.S. Pat. No.5,153,132, issued to Goodwin et al., and entitled "Three DimensionalCo-Culture Process, is closely related to this group of patents and isalso incorporated herein as if set out verbatim.

These prior patents disclose apparatuses that use either an internalcylindrical oxygenator or filter, as illustrated in FIG. 3, or a flatdisk shaped oxygenator membrane inserted internally between the walls ofthe vessel, as illustrated in FIGS. 1 and 2 in cross section andelevation, respectively. Both types of vessels require oxygen transferin order to sustain the growing cells.

Specifically, U.S. Pat. Nos. 4,988,623, 5,153,133, and 5,155,034disclose culture vessels that allow three dimensional cell growth. Thesevessels are shaped similarly to each other due to a central tubularmember that may either be an oxygenator that performs gas exchangethrough its surface, or it may be a cylindrical filter that allows thepassage of an oxygen rich culture medium. The presence of a centralizedtubular oxygenator or filter within a rotating vessel is a significantobstruction that will impede or prevent the growth of largerthree-dimensional cellular aggregates. Further, in cases where acylindrical filter has been used, it has been common to use internalblade members rotating about the central horizontal axis to move thefluid medium within the culture vessel. The use of such blades formixing the culture medium has been found to be unneccessary and evendetrimental due to the fluid shear that is caused by their rotation.

U.S. Pat. No. 5,153,131 discloses a bioreactor vessel without mixingblades or a central tubular membrane. This apparatus requires transferof gases into the bioreactor vessel. As shown in the cross sectionalview in FIG. 1, air travels through an air inlet passageway, through asupport plate member, across a screen, and through a flat disk permeablemembrane wedged between the two sides of the vessel housing. The oxygenthen diffuses across the membrane into the culture chamber due to theconcentration gradient between the two sides of the housing.

The rate at which oxygen can diffuse across the disk shaped membrane isa significant limitation that restricts the size of the culture chamber.Another disadvantage of the flat disk membrane in the '131 patent isthat it is designed to flex in order to cause mixing within the culturechamber. This mixing effect is a feature that is described as beingcritical for the distribution of air throughout the culture media,however, it will also tend to create shear within the chamber.

Consequently, an improved apparatus and method for suspending particles(cells and their substrate) that minimizes fluid turbulence, while atthe same time providing the required oxygen transfer, is needed toimprove the performance of bioreactors. More specifically, there is aneed for a bioreactor or culture vessel that provides sufficientinternal space to grow large three-dimensional cellular aggregates whileat the same time providing sufficient nutrients and gas exchange tosustain cell respiration and growth.

Providing sufficient gas exchange to sustain the growth of largercellular structures is a significant restriction when designing abioreactor or culture vessel. Attempts to overcome this problem havebeen directed in part at the use of gas permeable materials used to makethese reactors. For instance, U.S. Pat. No. 5,702,941, issued to Schwarzet al., and entitled "Gas Permeable Bioreactor And Method Of Use"discloses a vessel that is horizontally rotated and is at leastpartially composed of gas permeable materials. The gas exchange with theculture medium is intended to occur directly through the gas permeablematerials of which the vessel walls are composed. However, thespecification of the '941 patent notes that the range of sizes for thevessel is still limited since gas exchange is dependent on the quantityof gas permeable surface area. It is emphasized that although thesurface area of the vessel increases with the square of the dimensions,the volume of the vessel and thus, the internal culture medium,increases with the cube of its dimensions. As such, the preferreddimensions of the vessel described in the '941 patent are limited tobetween one and six inches in diameter while the width is preferablylimited to between one-quarter of one inch and one inch. Such sizelimitations are not suitable for growing three-dimensional cellularaggregates and tissues.

Similarly, U.S. Pat. No. 5,449,617, issued to Falkenberg et al., andentitled "Culture Vessel For Cell Culture" discloses a vessel that ishorizontally rotated. The vessel is divided by a dialysis membrane intoa cell culturing chamber and a nutrient medium reservoir. Gas permeablematerials are used in the vessel walls to provide gas exchange to thecell culturing chamber. However, the vessel is not completely filledwith the nutrient medium and a large volume of air is maintained abovethe fluid medium in both chambers. The vessel is not designed tominimize turbulence within the cell culture chamber, rather mixing isrecited to be essential to keep the dialysis membrane wetted. Further,the disclosure of the '617 patent does not contemplate using the vesselto grow cellular aggregates or tissues of any kind.

SUMMARY OF THE INVENTION

In the present invention, a cylindrical vessel with first and second endwalls defines a cell culture chamber which is rotatable about anapproximate horizontal axis. The first and second end walls are providedwith an inlet and an outlet respectively for introducing an oxygen richnutrient medium into and removing the spent medium and waste productsfrom the vessel. Filters are located near the inlet and the outlet toprevent the passage of cells and cellular aggregates from the culturechamber while allowing the passage of the nutrient medium and cellularmetabolic waste. The culture chamber is free of internal obstructionsand structures that might cause fluid shear or that might otherwiseimpede the growth and/or suspension of large three dimensional cellularaggregates while the vessel is being rotated. A pump provides a constantflow of the nutrient medium through the chamber while the vessel isrotated and a gas exchange device is used to transfer gases into and outof the nutrient medium.

In another aspect of the present invention, the operation of thebioreactor system is automated by providing means for controlling thetemperature of the vessel and/or nutrient medium. In addition, sensorsmonitor the flow rate, content, pH and temperature of the nutrientmedium as well as the rate of rotation of the vessel. A microprocessingunit records data from such sensors and directs adjustments formaintaining the desired operation conditions within the culture chamber.

In another aspect of the present invention, additional filters may beused in the cylindrical vessel intermediate between the end walls todefine or subdivide the culture chamber. Subdivision of the chamber isuseful for growing different types of cells and cellular aggregates, forcarrying out various cellular operations and functions and forconducting diagnostic research within the same cylindrical vessel. Whenthe culture chamber is subdivided into multiple sub-chambers, accessports may be provided along the cylindrical wall of the vessel toprovide access to each sub-chamber.

In another aspect of the present invention, the filter upstream relativeto the direction of flow of nutrient medium through the culture chambercan be cylindrical in shape and located adjacent to but spaced apartfrom the cylindrical wall of the vessel so that fresh nutrient mediumcan enter the culture chamber and/or sub-chambers at all points alongthe length of the vessel. The filters of the present invention are madeof a variety of materials and constructions and are chosen according tothe application for which the vessel is to be used.

It is an object of the present invention, to provide a method forculturing cells and growing three dimensional cellular aggregates andtissues. The method includes the steps of filling a rotatablecylindrical vessel having a culture chamber with an unobstructedlongitudinal axis with an oxygen rich fluid culture medium andintroducing cells, cellular aggregates and/or tissues into the medium. Aflow of oxygen rich fluid culture medium is maintained through thevessel to provide oxygen and materials to sustain cell respiration andgrowth and to remove cellular metabolic waste. The vessel is rotatedabout its horizontal longitudinal axis to suspend the growing cells andcellular aggregates in the medium. Periodically, the rotation of thevessel may be interrupted for the purpose of removing bubbles that mayhave formed in the nutrient medium and/or to remove materials from theculture chamber.

It is another object of the present invention to provide a method forfiltering biological waste materials from a fluid medium using tissuesor cellular aggregates to filter the waste materials from the medium.The method includes the steps of providing a rotatable cylindricalvessel having an interior chamber that is unobstructed along itshorizontal longitudinal axis. The chamber is filled with an oxygen richfluid culture medium and tissues and/or organoids are introduced intothe medium. As used in this disclosure, the term organoid refers to amass or aggregate of cells that mimics the structure and/or function ofa tissue or organ. A flow of oxygen rich fluid culture medium ismaintained through the vessel to sustain the organoids and to removecellular metabolic waste. The organoids are suspended in the medium bythe rotation of the vessel. A fluid containing the waste materials isthen passed through the chamber and the organoids remove the waste byvarious cellular mechanisms. Additional cylindrical vessels can beconnected in series to form a continuous chain of rotating vessels.Periodically, each vessel is replaced with another vessel containingfresh organoids.

It is yet another object of the present invention to provide abioreactor vessel that is disposable. Due to the present bioreactor'ssimple design and construction, it can be easily and economicallymanufactured. The resulting bioreactor is consequently affordable,disposable, and may be mass produced. In situations where minimizationof contamination is necessary, disposability of the bioreactor is aparticular advantage. While the bioreactor may be produced in a widevariety of sizes, its simple construction provides the added advantageof allowing the reactor vessels to be made smaller than previouslypossible. The smaller sizes are particularly useful in researchlaboratories. In the alternative, the features of the bioreactor systemof the present invention enable the growth of cells and cellularaggregates in larger volumes of media than was possible with prior artvessels. These larger volumes are particularly useful in the commercialscale production of biological materials.

Further aspects and objects of the present invention include providing adevice and method for growing cells and tissues for replacement ofdefective and damaged tissues in humans and other animals, providing adevice and method for growing cells and tissue cultures for theproduction of biological products such as but not limited to growthfactor proteins, enzymes, platelets and genetically engineeredmaterials, and providing a device and method for growing cells andtissue cultures for diagnostic procedures such as identifying andtesting chemotherapy and other biochemical agents.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above-recited features, advantages andobjects of the invention, as well as others which will become apparent,are attained and can be understood in detail, more particulardescription of the invention briefly summarized above may be had byreference to the exemplary preferred embodiments thereof which areillustrated in the appended drawings, which form a part of thisspecification. It is to be noted, however, that the drawing illustrateonly typical preferred embodiments of the invention and are thereforenot to be considered limiting of its scope as the invention may admit toother equally effective embodiments.

In the Drawings

FIG. 1 is a partial cross sectional view of a reactor vessel known inthe prior art showing the size limitation of such devices.

FIG. 2 is an elevated view of the prior art reactor vessel illustratedin FIG. 1, as mounted on a base having rotation means and gas exchangemeans.

FIG. 3, is also a perspective view of a prior art device that has anelongated growth chamber, but which provides oxygen injection through acentrally disposed cylindrical membrane.

FIG. 4, is a flow diagram illustrating the fluid flow in an apparatus ofthe present invention.

FIG. 5, is a cross sectional view of a reactor vessel of the presentinvention wherein the first filter is incorporated into the inlet.

FIG. 6, is a cross sectional view of a reactor vessel of the presentinvention wherein the first filter is attached to the first end wall.

FIG. 7, is a cross sectional view of a reactor vessel of the presentinvention wherein the culture chamber is defined by a cylindrical filteradjacent to but spaced apart from the cylindrical wall of the vessel.

FIG. 8, is a cross sectional view of a reactor vessel of the presentinvention wherein the culture chamber is defined by a cylindrical filterand is subdivided into sub-chambers by additional filter elements.

FIG. 9, is a cross sectional view of a reactor vessel of the presentinvention wherein multiple outlets are located about the periphery ofthe culture chamber.

FIG. 10, is a cross sectional view of a reactor vessel of the presentinvention wherein the downstream filter is adjacent to but spaced apartfrom the second end wall.

FIG. 11, is a cross sectional view of a reactor vessel of the presentinvention having a cylindrical filter along the length of the vessel anda downstream filter that is adjacent to but spaced apart from the secondend wall of the vessel.

FIG. 12, is a cross sectional view of a reactor vessel of the presentinvention that is particularly useful for filtering waste materials andtoxins from a fluid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to FIG. 4, in the overall system illustrated, a main fluidflow loop for growing mammalian cells includes a rotating cell culturereactor vessel 10, a gas exchange device or oxygenator 5, a main pump 3,and a supply manifold 1 for the selective input of nutrients, acids,bases, buffers and fresh medium to the circulating fluid nutrientmedium.

In a preferred embodiment of the invention, reactor vessel 10 is made ofa cylindrical vessel with first and second end walls, multiple filterelements and an unobstructed horizontal longitudinal axis defining anunobstructed culture chamber. The vessel has an inlet and an outlet, oneor more vessel access ports for transferring materials into and out ofthe vessel as well as means for removing bubbles from the nutrientmedium. The bioreactor system will have pump 3 for maintaining a flow ofnutrient medium through the vessel, gas exchange device 5 for dissolvinggases into and removing waste gases from the nutrient medium, and meansfor rotating the vessel about its horizontal axis. The bioreactor mayalso be provided with temperature control means, various sensors formonitoring the operational conditions and a microprocessor unit (notshown) for automating the operation of the bioreactor system.

Referring to FIG. 5, materials used to construct cylindrical wall 16 ofvessel 10 will preferably be a transparent, non-toxic, biocompatiblematerial such as glass or a clear plastic. Most preferably, the clearmaterial is a polycarbonate such as LEXAN® (a registered trademark ofGeneral Electric). The end walls will preferably be a material that isboth durable and machines well. Most preferably, the end walls will bemanufactured from an acetal polymer such as DELRIN® (a registeredtrademark of E. I. du Pont Nemours & Co., Inc.). Cylindrical wall 16 andend walls 12 and 14 may be formed by injection molding, wherein variousparts of the bioreactor vessel 10 may be welded, glued or mechanicallyattached together.

End walls 12 and 14 and cylindrical wall 16 may also be gas permeable.When desired, these structures may be made of a variety of materialssuch as silicone rubber, polytetrafluroethylene, polyethylene, porousplastic coated with a hydrophobic material, mixtures of silicone withother plastics, and silicone rubber coated cloth. When gas permeabilityis desired, preferably, the cylindrical wall 16 is constructed of aporous plastic coated with a hydrophobic material on the interiorsurface. Most preferably, the cylindrical wall 16, is made of poroushydrophobic Teflon® (a registered trademark of E. I. du Pont Nemours &Co., Inc.) when gas permeability is desired.

The cylindrical wall 16 may be made in any size provided that adequatenutrients and gases can reach the growing cells and cellular aggregateswithin the culture chamber. The size of the vessel is therefore limitedonly by the flow rate of the fluid nutrient medium into the culturechamber and the contents of that medium. The prior art discloses reactorvessels that have a culture chamber with a length of at least 0.25inches but no more than 1 inch. The preferred volumes of these prior artvessel chambers is generally less than 500 ml. However, when sufficientnutrient flow is provided through the culture chamber within vessel 10of the present invention, the length and diameter of cylindrical wall 16will be much larger than the dimensions of these prior art bioreactors.More specifically, because the culture chamber of the present inventionhas maximum dimensions that are determined by the flow rate and contentof the fluid medium that passes through the culture chamber, the culturechamber can have maximum lengths well in excess of 1 inch and can haveinternal volumes up to 100 liters.

The end walls of vessel 10 include first end wall 14 and second end wall12. The nutrient medium flows through the vessel generally in only onedirection and as such, the ends of the vessel are periodically referredto as upstream and downstream, respectively. However, as noted below,the flow of the nutrient medium may be periodically reversed, and thus,the end walls are more generally referred to as first and second endwalls.

In FIG. 5, first end wall 14 is connected to the upstream end ofcylindrical wall 16 to form the upstream portion of vessel 10. Thecylindrical wall and the two end walls may be glued, welded ormechanically attached to one another to form vessel 10. First end wall14 is provided with inlet 20 that is coupled via rotative coupling 22 toconduit 24. First end wall 14 is shown with only a single inlet 20.However, it is anticipated that conduits may be machined into end wall14 to provide additional inlets into the culture chamber at variouslocations on the end wall. More specifically, radially oriented boresmay machined into the end wall so that a plurality of inlets is arrangedabout the periphery of end wall 14. Such an arrangement of inletsprovides a greater distribution of the oxygen rich nutrient medium as itenters the culture chamber through end wall 14.

The inlet or inlets of end wall 14 are in fluid communication withconduit 24 which is in fluid communication with gas exchange device 5.As shown in FIG. 4, main pump 3 provides fresh nutrient medium to thegas exchange device wherein the nutrient medium is oxygenated and passedon to vessel 10. The return spent nutrient medium from vessel 10 isreturned to manifold 1 where it receives a fresh charge of nutrients,acid, base, buffer, or liquid medium, as necessary before recycling.Adjustments to the nutrient medium may be made in response to chemicalsensors suspended in the medium and/or to electrochemical sensorslocated within or down stream of vessel 10. The pH of the medium iscorrected by controlling carbon dioxide pressures and introducing acids,bases and/or buffers. Dissolved oxygen, nitrogen and carbon dioxide aremaintained in appropriate concentrations by gas exchange device 5 inorder to support cell respiration. Spent medium may be directed to awaste or drain as it passes from the vessel. Alternatively, where thecells, cellular aggregates and/or tissues within vessel 10 aresynthesizing and/or excreting materials that are to be retained, thosematerials may be directed to collection device 11.

Referring again to FIG. 5, first end wall 14 is provided with accessport 26 for accessing the interior of the culture chamber. Although onlya single port 26 is shown in the figures, additional access ports may beutilized depending on the particular application of the bioreactor. Asshown in FIG. 5, the access port 26 provides access to the vessel forthe input of medium and cells and for the removal of cultured cells andcellular aggregates. In the preferred embodiment, the vessel accessports are constructed with valved closure means or a septum-membraneclosure. The valves preferably are plastic, but may be made of metal orany other material which is non-toxic, capable of being sterilized andis hard enough for machining into an access port. Further, access port26 may be provided with a variety of couplings for connecting withvarious fittings. Where the culture chamber within vessel 10 issubdivided into sub-chambers, access ports may be provided along thelength of the cylindrical wall 16 in order to allow access to each ofthe various sub-chambers.

Vessel 10 is also provided with means for trapping bubbles that maydevelop in the culture chamber and an adjacent port is provided forremoving the bubbles from the bubble trapping means. As shown in FIG. 5,bubble removal port 28 is incorporated into first end wall 14. Althoughnot shown, bubble trapping means may take a variety forms. For instance,a portion of the inner surface of cylindrical wall 16 may be providedwith a recessed structure that is designed to trap bubbles during therotation of vessel 10. Regardless of the structure that is used to trapbubbles, an access port such as bubble removal port 28 should be locatedadjacent to such means. Further, it is anticipated that bubble removalport 28 will have a valve type closure so that a hypodermic syringe maybe attached to remove bubbles from the chamber so as to minimizeturbulence that might be created during the removal of bubbles.Alternatively, a septum membrane type closure may also be employed.

Second end wall 12 is connected to cylindrical wall 16 to form thedownstream portion of vessel 10. Second end wall 12 is provided withcentral outlet 18 and is connected to drive shaft 32. Drive shaft 32 isconnected to means for rotating the shaft and the vessel as is discussedbelow. Alternatively, it is anticipated that means for rotating thevessel may be directly attached to either end wall or the cylindricalwall of the vessel. However, the balance of this description assumesthat the rotation means is adapted to rotate the drive shaft 32 which isfixedly connected to the second end wall 12. Central outlet 18 is influid communication with conduit 58 for directing fluid medium away fromthe vessel. Second end wall 12 may have only central outlet 18 or it mayhave a plurality of outlets 48. As shown in FIG. 9, outlets 48 may bearranged about the periphery of vessel 10 with fluid communication withcentral outlet 18 provided via wall bores 60. Plurality of outlets 48can have a variety of configurations as determined by the particularapplication of the bioreactor and the ease of manufacture.

Since the operation of the bioreactor of the present invention requiresa constant flow of oxygen rich fluid nutrients through the vessel,filters are used define and subdivide the culture chamber and to retainthe growing cells, cellular aggregates, tissues and/or organoids in theculture chamber. Therefore, the use of filters is preferred overdialysis membranes and the like. The filter should be of a size thatallows the passage of sufficient oxygen rich nutrient medium to sustaincell respiration and growth while preventing the passage of cells,cellular aggregates tissues and/or organoids. The combination of such afilter with a constant flow of oxygen rich medium will sustain cellularrespiration and growth within a much larger vessel than was previouslyknown. Further, by providing a continuous flow of oxygenated nutrientmedium, it is no longer necessary to periodically change the nutrientmedium, a process that interrupts the rotation of the vessel andincreases the risk of contaminating the culture or otherwise damagingthe cells, cellular aggregates, tissues and/or organoids.

The filters used in the present invention may be made of a variety ofmaterials having a variety of constructions provided that they have aporosity that allows the nutrient medium and cellular metabolic waste totravel through the filter but that will prevent the passage of cells andcellular aggregates. In particular, the filters of the present inventionmay be made of a polycarbonate film that has been irradiated to renderit porous, polyester cloth and various woven materials such as a wovenfabric of stainless steel. Such filters are commercially available froma variety of sources.

It is anticipated that filters made of biodegradable materials such aspolyglycolic acid may also be used. In particular, there will beapplications when the growth of cellular aggregates, tissues and/ororganoids will either require a substrate or the use of a substrate willprovide a desired shape or structure in the grown product. For suchapplications, a filter made of biodegradable material that will slowlydegrade after the growing cells have attached to the upper surface ofthe filter is desirable.

The size, mesh and location of the filters can vary widely. The meshsize of the filter material or construction is determined in large partby the application for which the bioreactor system is to be used. If thereactor vessel is used to produce or test white blood cells the meshsize will need to be quite small to prevent the leukocytes from passing.If the vessel is being used to produce a bone marrow, a larger mesh sizewill be sufficient. Culture work concerning the production or testing ofcellular aggregates such as tissues and organoids can utilize stilllarger mesh sizes.

A first filter 34 should be used to prevent the cells and cellularaggregates in the culture chamber from travelling upstream from thereactor should the fluid flow through the reactor be reversed. Asillustrated in FIG. 5, filter 34 can be a small filter fixed withininlet 20 or as shown in FIG. 6, can be arranged across the full diameterof the vessel attaching to end wall 14 and cylindrical wall 16 at itsperiphery. In addition, the filter elements may be sandwiched betweenthe various components of vessel 10 such as between the end walls andthe cylindrical wall as is shown in FIG. 12.

To improve the distribution of the nutrient medium to the culturechamber, filter 34 may have additional configurations.

For instance, filter 34 may be attached about its periphery to thecylindrical wall 16 of the vessel, adjacent to but spaced apart from endwall 14 as shown in FIG. 12. In such a configuration, the nutrientmedium enters vessel 10 through inlet 20 and passes across filter 34 atall points on the filter such that an improved distribution of medium isachieved.

Additionally, as illustrated in FIG. 7, the upstream filter may beformed into cylindrical structure 40 and attached to downstream filter36 to form a filter enclosed culture chamber 30. The particularadvantage of cylindrical filter 40 is that the nutrient medium passesbetween cylindrical wall 16 and filter 40 and can thus pass through thefilter directly into all parts of culture chamber 30 along the length ofthe vessel. As such, the nutrient medium is not required to pass througha series of chambers and/or filters to reach the downstream portions ofculture chamber 30 or downstream sub-chambers. Alternatively, the flowof medium may be directed immediately into culture chamber 30 and outthrough cylindrical filter elements 40 as shown in FIG. 11. With such aconfiguration, upstream filter element 20 may be unnecessary in that itis unlikely that the cylindrical filter element 40 and downstream filterelement 46 will simultaneously become clogged such that any backflushing would be required.

FIG. 8 illustrates how the culture chamber may be subdivided intosub-chambers 30a, 30b and on out to 30n, where n is a positive integergreater than 1, by intermediate filter elements 42. Again, the advantageof this configuration is that nutrient medium can pass betweencylindrical wall 16 and cylindrical filter 40 and provide fresh nutrientmedium to each sub-chamber along the length of the vessel. Although notillustrated in the figures, cylindrical wall 16 can have access portsalong its length to provide access to culture chamber 30 and tosub-chambers 30a-30n when the chamber is subdivided by intermediatefilter elements 42.

In addition to those filter elements disposed upstream of and withinculture chamber 30, a filter element downstream of the chamber is alsorequired to prevent the cells, cellular aggregates, tissues and/ororganoids from passing out of the vessel. Again, filter 36 can have avariety of configurations within and without vessel 10. As illustratedin FIGS. 5-6 and 7-8, filter 36 can be provided along the surface ofsecond end wall 12. Alternatively, filter 36 may be arranged withinoutlet 18 (not shown) or attached about its periphery to cylindricalwall 16 and spaced apart from outlet 18 as illustrated in FIG. 10.

It is anticipated that filter 36 may periodically become clogged withcells and cellular aggregates. In such an instance, the direction of theflow of the nutrient medium should be reversed by pump 3 until the cloghas been cleared. The direction of flow of the nutrient medium may alsobe reversed in order to cause the suspended cells, cellular aggregatesand/or tissues to become attached to a substrate or the filter elementsof the vessel.

As illustrated in FIG. 4, the bioreactor system is provided with pump 3which maintains a flow of fluid nutrient medium through the vessel 10.Pump 3 may be a peristaltic pump or similar device that is capable ofmaintaining a relatively constant flow of fluid medium through thereactor vessel. Pump 3 may be adjusted to a variety of flow rates and iscapable of reversing the direction of medium flow through the vessel.

The bioreactor system is also provided with gas exchange device 5. Gasexchange device 5 may be characterized as an oxygenator, but the deviceshould be capable of maintaining desired gas concentrations for thevariety of gases needed to sustain and promote cellular respiration.Oxygen is consumed in the culture chamber and carbon dioxide is givenoff as a byproduct of cellular respiration. Thus, the gas exchangedevice must be capable of transferring oxygen into and removing carbondioxide from the nutrient medium. If not properly balanced, theincreasing quantities of carbon dioxide will render the circulatingmedium acidic.

In the gas exchange device used in the present invention, gases aretransferred into and out of the nutrient medium across a multi-layeredcylindrical membrane composed primarily of silicone rubber. Thecylindrical membrane is located in a cylindrical housing that hasupstream openings for receiving the nutrient flow from the pump anddownstream openings for passing the oxygen rich medium onto the reactorvessel. The housing is provided with a fan attached thereto to maintaina constant flow of air through the housing and across the surfaces ofthe cylindrical membrane.

The present invention involves the rotation of reactor vessel 10 aboutits central horizontal axis. This involves a type of clinostatprincipal, i.e. a principal that fluid rotated about a horizontal ornearly horizontal axis can effectively suspend particles in the fluidindependent of the effects of gravity. The rotational speed of vessel 10effectively eliminates the velocity gradient at the boundary layerbetween the fluid and cylindrical wall 16. Thus, shear effects causedbetween a rotating fluid and stationary wall are significantly reducedor eliminated.

The clinostat principal involved allows cells or cell aggregates havingdensities different from the fluid to travel in a nearly circular pathand to deviate insignificantly from the fluid path. Relative to therotating reference frame, the gravity vector is observed to rotate sothat its average time is nearly zero. This allows for suspension of theparticles in a carrier medium with low fluid shear and with lowinterference. Cylindrical wall 16 is rotated in order to reduce theadverse fluid velocity gradient through the boundary layer that wouldotherwise occur at the interface between the moving fluid and the fixedwall. The rotation of cylindrical wall 16 is sufficient to cause fluidrotation due to viscosity. The operating limits are defined by thesedimentation rate of the particles in the fluid medium and theacceptable centrifugal force due to rotation. Further, it is possible tovary the angular rotation rate in order to induce secondary flowpatterns within the vessel which may be useful for distributingnutrients or waste products.

In the present invention, vessel 10 is rotated about a horizontal axisand the process utilizes zero head space of fluid medium within thevessel. The zero head space results in no air bubbles which might causedisruption of the fluid streamlines and thereby subject the culture toadverse shear effects. The preferred means for rotation is a motorassembly (not shown). The motor assembly is fixed to mounting base andis provided with means for attaching to and rotating vessel 10. Forinstance, attachment means may comprise threadably connecting the vessel10 to the motor assembly 54 through screw threads on drive shaft 32corresponding to screw threads on end wall 12 of vessel 10. These screwthreads are in a direction such that inadvertent loosening of vessel 10from the motor assembly 54 due to the movement of rotation is avoided.In addition, a lock nut or similar device may be provided on the driveshaft to prevent unscrewing. However, it is preferred that theattachment means be a series of sprocket gears that cause drive shaft32, and vessel 10 fixedly attached thereto, to rotate about itshorizontal axis.

The means for rotation in yet another embodiment is a roller mechanismhaving multiple rollers arranged longitudinally in a horizontal plane.The rollers are rotated simultaneously to rotate a reactor vessel laidbetween the rollers. Such a roller mechanism may be obtained from StovalLife Science, Inc. but other roller mechanisms that will providecontrolled rotation may also be used.

The preferred speed of rotation is in the range of about 2.0 revolutionsper minute (rpm) to about 45 rpm. The desired speed of rotation isdependent on the specific dimensions of the vessel 10 and the particularapplication. For example, for a bioreactor of about 3 to about 5 inchesin diameter, with a width of about 0.25 inches, growing BHK-21 cells ina microcarrier culture, the preferred speed of rotation is about 24 rpm.However, in vessels having larger dimensions, the preferred speed ofrotation will be decreased to perhaps about 10 to about 15 rpm. It is tobe anticipated that the speed of rotation must be adjusted to balancethe gravitational force with the centrifugal force caused by thatrotation, particularly as larger diameter vessels are used. Further, ascellular masses suspended in the culture medium increase in density withcellular growth, increased rotation rates will be required to maintainthose masses in a suspended state.

While the rotation of the vessel 10 may take place by rotating thevessel about the substantially longitudinal central axis in asubstantially horizontal plane, it may also take place by rotating thevessel in a plane inclined no more than about 10 degrees from asubstantially horizontal plane.

This inclination may become necessary where the flow of medium throughthe culture chamber is sufficient to carry the growing cells and/ortissues toward the downstream portion of vessel 10.

Vessel 10 may be provided with temperature control means so as tocontrol the temperature of medium within the vessel and the temperatureof the medium within the reservoirs 13, 15 and 17 as shown in FIG. 4. Inthe alternative, the entire bioreactor system may be operated within anenlarged incubator to maintain all of the bioreactor system elements ata desired operating temperature. The desired temperature will bedetermined by the particular application of vessel 10 and the types ofmaterials being grown therein.

The bioreactor system of the present invention may also be constructedwith means for attaching vessel 10 to additional similar vessels,thereby creating a chain or series of bioreactors. The vessels in such achain are connected to one another by attachment means located on theirrespective end walls and/or drive shafts. When a chain of bioreactors isformed in this manner, the chain may be attached to a means for rotationat one of its ends for rotation. If a motor assembly is used forrotation of the chain of reactor vessels, access ports 26 should belocated on the cylindrical wall 16 of the vessels for easier access.However, if the chain of bioreactors is to be laid on a roller mechanismfor rotation, the vessel access ports 26 should be located on the endwalls of the vessels.

Another aspect of the present invention is a method for growing cells,cellular aggregates and/or tissues in a bioreactor system comprisingfilling a vessel, having an unobstructed horizontal longitudinal axis,with a liquid culture medium. Cells, cellular aggregates and/or tissuesare suspended in the liquid culture medium and the vessel is rotatedabout its horizontal longitudinal axis at a rate that suspends the cellsin the liquid nutrient medium. A flow of oxygenated nutrient medium ismaintained through the vessel to sustain cellular respiration andgrowth. The rotation of the vessel and the flow of medium is maintainedfor a period of time to attain desired cell and/or tissue growth.

More specifically, after sterilization, vessel 10 is filled with a fluidnutrient medium, such as those commonly known in the art for growingcells and cellular aggregates, and cells; The nutrient medium mayinclude a variety of materials to sustain the cells, to promote thegrowth of certain cells, and/or to promote the production or excretionof certain substances by the cells and/or tissues. These materials mayinclude fetal bovine serum, regulatory proteins, salts, sugars,dissolved gases and other materials that combine to form a fluidnutrient medium that approximates blood plasma. Substrate particles suchas collagen coated beads and the like may be added to the medium ifdesired. Further, tissue explant material may be diced and added to themedium either as a substrate for growing other cellular materials or asthe as the culturing material for further cell growth.

Once vessel 10 is completely filled with the medium so that no airspaces exist in the vessel, the cells and/or tissues are introduced intothe medium and the vessel is rotated as described above. The rate ofrotation will depend on the volume of the culture chamber of the vesseland the density of the growing mass of cells and/or tissues. Forresearch purposes, the volume of culture vessel 10 may range from 55 ml.to about 500 mls. For commercial purposes, the volume of the culturevessel may range from about 100 ml to as much as 100 l. As the densityof the growing cells will increase with growth, the rate of rotationwill likely need to be periodically adjusted to compensate for suchchanges.

As the materials of cylindrical wall 16 are preferably transparent, thegrowth of the cells and/or cellular aggregates may be visuallymonitored. The length of time over which the bioreactor system isoperated will vary greatly depending on the application to which thesystem is being used. When the system is used for diagnostic purposes,vessel may be rotated for only a matter of hours. However, when tissuesand large cell masses are to be grown or the secretions of such tissuesand/or masses is to be produced, a vessel may be operated over a periodof days, weeks or even months.

A continuous flow of oxygen rich nutrient medium is supplied to vessel10 while the cell, cellular aggregates, tissues and/or organoids aresuspended by the rotation of the vessel. The flow rate of the nutrientmedium is critical since the culturing of cells and tissues requires aminimum supply of nutrients and oxygen to sustain respiration. The flowrate of the medium depends on the size, and thus, the volume of theculture chamber within the vessel. A higher flow rate being required toprovide sufficient oxygen and nutrients to a larger chamber. It isanticipated that the flow of medium into the vessel may be as high as 10ml/min. However, at this flow rate the filter elements of the vessel arelikely to clog and periodic reversing the direction of medium flow maybe required to back flush the clog from the filters. In the preferredmethod, the flow rate will vary between 2 and 3 ml/min.

Temperature control is also essential to maintaining culture chamber 30at an optimum temperature for cell growth. Preferably, the desiredtemperature will be maintained by operating the entire bioreactor systemwithin an incubator. In the alternative, temperature control means maybe utilized in the medium reservoirs and within vessel 10. Thetemperature will preferably range from about 35° C. to about 40° C. formammalian cells.

When the desired level of growth or production has been reached,rotation is stopped, the vessel access ports are opened and the cellularmaterials removed. If vessel 10 is made of sterilizable materials thevessel may be emptied and sterilized for future use. If disposable, thevessel and any undesirable contents are destroyed.

Another aspect of the present invention is to provide a method forfiltering waste materials from fluids. As shown in FIG. 11, organoids ofliver, kidney, pancreatic and other tissues and/or cells may besuspended in the rotating culture chamber 30. A fluid containing wastematerials or toxins is added to the fluid nutrient medium passing intothe reactor vessel and through filter element 34. As the fluid mediumpasses through the culture chamber, the organoids suspended in thechamber filter the waste material from the fluid utilizing variouscellular mechanisms. The cellular mechanisms carried out by theseorganoids are the same as or similar to the mechanisms used by humanorgans to remove toxins and other waste materials from the human body.

There is a limit to the quantity of materials that can be removed fromthe fluid flowing through a single reactor vessel. As the ability of thesuspended organoids to filter toxins becomes exhausted, the organoidswill frequently expire. Therefore, it is preferable that the bioreactorsystem will have multiple vessels arranged in series so thatperiodically, a vessel containing depleted organoids may be removed andreplaced with a vessel containing fresh organoids. The fluidcommunication between the vessels in such a series allows for moreefficient filtering and further allows the filtering process to continuewithout significant interruption when the organoids in a vessel need tobe replaced.

In another aspect of the invention, the reactor vessel of the presentinvention may be used as a method of carrying out various diagnosticprocedures. By way of example only, the reactor vessel of the presentinvention may be used to test new chemical agents for treating variousdiseases or cancers. In particular, a reactor vessel that has beensubdivided into a number of sub-chambers may contain diseased orcancerous tissues in one sub-chamber while liver, kidney and othertissues may be suspended in the other sub-chambers. The fluid mediumcontaining a chemical agent for treating the diseased tissues iscirculated through the vessel for a desired period of time to determinethe affects the agent. The significance of this procedure is that theaction of the agent on the diseased or cancerous tissue may be monitoredin the presence of various organ tissues that may counteract the affectsof the agent or that may be adversely affected by the agent.

As noted, the bioreactor system of the present invention can be used fora variety of applications. In terms of research, the reactor vessel maybe used in researching cancer, HIV, tissue modeling, genetics, tissuemaintenance, virology, extracellular matrix interactions, signaltransduction and protein discoveries. In terms of the tissueregeneration, the reactor vessel can be used to generate bone marrow,liver, pancreas, skin, heart, nerve, cartilage, kidney, blood and bloodvessel tissues. Likewise, the reactor vessel may be used to producevarious tissues, pharmaceuticals, diagnostic agents, vaccines, cellularaggregates and organoids.

Cells and tissues that can be grown in the bioreactor system of thepresent invention include human keratinocytes, epithelial and fibroblastcells of small intestine, lymphocytes, melanocytes, embryonic cells,osteoblasts, hepatocytes, bone marrow and bone marrow stem cells.Various cancers that can be produced in the reactor vessel of thepresent invention include neuroblastoma, breast, prostate, lung,melanoma, kidney, and ovarian cancers and adenocarcinoma. Examples ofviruses that can be grown in the reactor vessel of the present inventioninclude AIDS/HIV, ebola, HHV8-Kaposi's Sarcoma, influenza, Epstine Barrvirus, Monkey pox and Norwalk.

From the foregoing it will be seen that this invention is one welladapted to attain all of the ends and objects hereinabove set forth,together with other advantages which are obvious and which are inherentto the apparatus and structure.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations. This is contemplated by and is within the scope of theclaims.

Because many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A method for filtering biological waste materialand toxins from a fluid medium using cellular filtering, comprising thesteps of:providing a rotatable cylindrical vessel having a filterchamber with an unobstructed longitudinal axis; filling the vessel withan oxygen rich fluid culture medium; introducing live organoids into themedium; maintaining a flow of oxygen rich fluid culture medium throughthe vessel to provide oxygen and materials to the living organoids andto remove cellular metabolic waste; rotating the vessel to suspend theorganoids in the medium; and passing a fluid containing a waste materialand toxins through the chamber wherein the suspended organoids removethe waste material and toxins from said fluid using cellular mechanisms.2. The method of claim 1, further comprising the step of providingadditional cylindrical vessels in series with fluid communicationbetween the vessels.
 3. The method of claim 2, further comprising thestep of periodically removing a vessel containing depleted organoidsfrom the series and adding a vessel containing fresh organoids to theseries.